Amino-derivatized phosphoramidite linking agents

ABSTRACT

The compounds of the invention include novel linking agents comprising 2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes and their phosphoramidite precursors. The compounds of the invention further include conjugates of the above linking agents with oligonucleotides and polymer supports. The compounds of the present invention are useful for linking organic moieties, such as fluorescent or chromogenic dyes, to polymer supports and oligonucleotides, particularly single- and double-stranded DNA and RNA fragments.

This is a divisional of co-pending application Ser. No. 06/769,170 filedon Aug. 26, 1985, now U.S. Pat. No. 4,757,141.

BACKGROUND

The invention relates generally to organophosphorous compounds, and moreparticularly, to organophosphorous compounds for synthesizingamino-derivatized polymers, especially oligonucleotides.

Genes and gene control regions can now be routinely characterized andstudied at the molecular level. This is possible because of severalrecent advances in the technology associated with manipulating andmodifying deoxyribonucleic acid (DNA). Of particular importance havebeen advances in DNA sequencing, Maxam and Gilbert, "SequencingEnd-Labeled DNA with Base-Specific Chemical Cleavages," and Smith, "DNASequence Analysis by Primed Synthesis," pgs. 499-560 and 560-580,respectively, in Methods in Enzymology, Grossman and Moldave, eds., Vol.65 (Academic Press, New York, 1980); the isolation of a large number ofhost restriction modification enzymes, Roberts, "Dictionary ofRestriction Endonucleases," in Methods in Enzymology, Wu, ed., Vol. 68(Academic Press, New York, 1979); and the construction of vectors forcloning and amplifying defined DNA sequences, e.g. Bolivar and Backman,"Plasmids of Escherichia coli as Cloning Vectors," in Methods inEnzymology, Wu, ed., Vol. 68 (Academic Press, New York, 1979).

Many of these new techniques require that DNA fragments oroligonucleotides be labeled or attached to polymer supports. DNAsequencing techniques and gene probes, which can be used to help locatenatural genes of commercial or scientific importance, require the use oflabeled oligonucleotides. Until recently, all DNA sequencing techniquesrelied on radioactive labels for distinguishing oligonucleotidefragments separated by electrophoresis. Radioactive labels are highlysensitive, and can be incorporated without steric hinderance, or otherchemical side effects. However, their use poses a laboratory healthhazard, which requires that they receive special handling and disposal.Moreover, their use is not amenable for rapid automatic sequencing ofoligonucleotides, as nucleoside-specific radioactive labels are notavailable for practical identification of different nucleotide bases,and radiation detection techniques such autoradiography andscintillation counting are too time consuming. As a consequence, othernon-radioactive labelingtechniques have been sought, such as fluorescentand colorimetric labeling, which depend on the ability to covalentlylink a fluorescent or chromogenic molecule to an oligonucleotide.

Chu et al, in "Derivatization of Unprotected Polynucleotides," NucleicAcids Research, Vol. 11, pgs. 6513-6529(1983), disclose a method forattaching amines to the terminal 5'-phosphates of oligonucleotides. Oneobject of the method is to provide a means for attaching organiclabeling molecules to oligonucleotides by way of an amine linkage. Themethod involves treating the oligonucleotides with a carbodiimide.

Chollet and Kawashima, in "Biotin-Labeled SyntheticOligodeoxyribonucleotides: Chemical Synthesis and Uses as HybridizationProbes," Nucleic Acids Research, Vol. 13, pgs. 1529-1541 (1985),disclose the use of the method of Chu et al to attach biotin to the5'-phosphate of an oligonucleotide. The reported yields of 50-70% arebelow that needed for use in automatic synthesizers, and thecarbodiimide can cause unwanted modifications to oligonucleotide basesin the course of the reaction.

Smith et al, in "Synthesis of Oligonucleotides Containing an AliphaticAmino Group at the 5' Terminus: Synthesis of Fluorescent DNA Primers forUse in DNA Sequence Analysis," Nucleic Acids Research, Vol. 13, pgs.2399-2412 (1985), disclose a protected amino-derivatized nucleosidephosphoramidite for linking fluorescent or colorimetric tags tooligonucleotide fragments. While the linker is highly useful forattaching base-specific labels to the 5' terminus of oligonucleotides,the protected-amine phosphoramidite is not readily purified.

Connolly and Rider, in "Chemical Synthesis of OligonucleotidesContaining a Free Sulphydryl Group and Subsequent Attachment of ThiolSpecific Probes," Nucleic Acids Research, Vol. 13, pgs. 4485-4502(1985), disclose the synthesis of oligonucleotides having atrityl-protected sulphur attached via a two, three, or six carbon chainto the 5' phosphate of the oligonucleotide.

Apart from linking labeling agents to oligonucleotides, there is greatinterest in immobilizing various molecules on polymer supports, such ascatalysts, enzymes, microorganisms, affinity reagents, immunoadsorbents,and the like, for both preparative and analytical uses, e.g. Schott,Affinity Chromatography (Marcel Dekker, Inc., New York, 1984), andMosbach, ed., Methods in Enzymology, Vol. 44, "Immobilized Enzymes,"(Academic Press, New York, 1976). Of particular interest in this fieldare means for immobilizing molecules and cells by covalent bonds.

SUMMARY OF THE INVENTION

The compounds of the invention include novel linking agents comprising2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes and theirphosphoramidite precursors. The compounds of the invention furtherinclude conjugates of the above mentioned linking agents witholigonucleotides, conjugates of the above mentioned linking agents withpolymer supports, and conjugates comprising dyes linked tooligonucleotides by the above mentioned linking agents. The presentinvention relates to compounds that are useful for linking organicmoieties, such as fluorescent and chromogenic dyes, to DNA fragments andoligonucleotides, particularly single-stranded DNA and RNA, and forlinking DNA fragments, oligonucleotides, proteins, and the like topolymer supports. The compounds and their conjugates are useful inautomated and manual DNA and RNA synthesis and sequence analysis,construction of gene probes, affinity techniques, and the like. Inparticular, the cyclic embodiments of the linking agents of theinvention advantageously overcome deficiencies associated with currentlyavailable linking methods by providing more readily purified linkingagents.

DESCRIPTION OF THE INVENTION

The linking compounds of the present invention include2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes defined by theformula: ##STR1## wherein:

n is in the range of 2 to 4, preferably in the range of 2 to 3, and mostpreferably is equal to 2.

R₁ represents an amino protection group, preferably either acid-labileor base-labile, e.g. as described by Greene, in Protective Groups inOrganic Synthesis (John Wiley & Sons, New York, 1981), chapter 7, whichchapter is incorporated by reference. Preferably base-labile protectiongroups when taken together with the nitrogen of the heterocycle or thatof its precursor, are base-labile amide and carbamate protection groups,preferably trihaloacetyls, acetoacetyl, and fluorenylmethyl carbamates,particularly 9-fluorenylmethyl carbamate and 9-(2-sulfo)-fluorenylmethylcarbamate, and most preferably trifluoroacetyl. Preferable acid-labileprotection groups include trityls, and their lower (containing from 1-3carbon atoms) alkoxy derivatives, particularly 4-monomethoxytrityl and4,4-dimethoxytrityl.

R₂ and R₃ are chosen so that (1) the likelihood that they stericallyhinder the cyclization of the compound of Formula I is minimized, (2)the ring electron density of the heterocycle of Formula I is reduced,because it is thought that this will enhance the reactivity or the N-Pbond in the compound of Formula I to hydroxyl groups, and (3) themolecular weight of the compound of Formula I is minimized to increasethe likelihood that it can be purified by distillation. R₂ and R₃ takenseparately each represent hydrogen, lower alkyl, lower substitutedalkyl, particularly halo-, cyano-, or nitro- substituted lower alkyl,lower acyl, cyano, halo, and nitro; more preferably R₂ and R₃ takenseparately each represent hydrogen, lower alkyl, and lower haloalkyl;and most preferably R₂ and R₃ represent hydrogens.

R₄ represents alkyl, alkenyl, aryl, aralkyl, or cycloalkyl containing upto 10 carbon atoms. More preferably, R₄ represents lower alkyl;electron-withdrawing beta-substituted ethyl, particularlybeta-trihalomethyl-, beta-cyano-, beta-sulfo-, beta-nitro-substitutedethyl, or the like; electron-withdrawing substituted phenyl,particularly halo-, sulfo-, cyano-, or nitro-, substituted phenyl; orelectron-withdrawing substituted phenylethyl. Most preferably, R₄represents methyl, beta-cyanoethyl, or 4-nitrophenylethyl.

The term "lower alkyl" as used herein denotes straight-chain andbranched-chain alkyl groups containing from 1-6 carbon atoms, e.g.methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl,neopentyl, tert-pentyl, and the like. "Lower substituted alkyl" denoteslower alkyl having electron-withdrawing substituents, such as halo,cyano, nitro, sulfo, or mono-, di-, or trihalomethyl, or the like."Lower haloalkyl" denotes a lower alkyl with one or more halogen atomsubstituents, usually fluoro, chloro, bromo, or iodo. "Lower acyl"denotes an acyl containing from 1-7 carbon atoms wherein the non-doublebonded carbons comprise a lower alkyl, possibly having halo-, cyano-, ornitro- substituents. "Electron-withdrawing" denotes the tendancy of asubstituent to attract valence electrons of the molecule of which it isapart, i.e. it is electronegative.

A special case of the above-described linking agent includes bicycliccompounds defined by the formula: ##STR2## wherein m is in the range of1 to 3, and n, R₁, and R₄ are as defined above. More preferably m is inthe range of 1 to 2; and most preferably m is 1. The lower cycloalkylattached to the oxazaphospha-heterocycle is though to introduce ringstrain into the heterocycle making the nitrogen-phosphorous bond morereactive.

The linking compounds of the invention also include the phosphoramiditeprecursors to the above2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes, the precursorsbeing defined by the formula: ##STR3## wherein:

R₁, R₂, R₃, and R₄ are as indicated above; j is in the range of 2 to 10,more preferably in the range of 2 to 4, and most preferably j is in therange or 2 to 3 (it is believed that this latter range results in aprecursor possessing the most favorable steric configuration forcyclization); and

R' and R" taken separately each represent alkyl, aralkyl, cycloalkyl,and cycloalkylalkyl containing up to 10 carbon atoms. Preferably R' andR" taken separately represent lower alkyl, and most preferably when theabove phosphoramidites are used directly as linking agents, R' and R"taken separately are sterically hindering lower alkyls which enhance thechemical stability of the phosphoramidites, and hence their shelf lives.Such sterically hindering lower alkyls include isopropyl, t-butyl,isobutyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, sec-pentyl, andthe like. Most preferably, when the above phosphoramidites are used asprecursors to the above-described oxazaphospha-heterocycle, R' and R"taken separately are isopropyl.

R' and R" taken together form an alkylene chain containing up to 5carbon atoms in the principal chain and a total of up to 10 carbon atomswith both terminal valence bonds of said chain being attached to thenitrogen atom to which R' and R" are attached; or R' and R" when takentogether with the nitrogen atom to which they are attached form asaturated nitrogen heterocycle which may contain one or more additionalheteroatoms from the group consisting of nitrogen, oxygen, and sulfur.More preferably, R' and R" taken together and with the nitrogen to whichthey are attached represent pyrrolidino, morpholino, or piperidino. Mostpreferably, R' and R" taken together and with the nitrogen to which theyare attached represent morpholino.

The phosphoramidite precursors to the above-described2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes includesubstituted lower cycloalkanes defined by the formula: ##STR4## whereinR₁, R₄, R', and R" are as indicated above, and t is in the range of 0 to8, and s is in the range of 0 to 8, subject to the condition that s+t isin the range of 1 to 8, more preferably t is in the range of 0 to 2, ands is in the range of 1 to 3, still more preferably t is 0 and s is inthe range of 1 to 2, and most preferably t is 0 and s is 1.

Conjugates of the present invention include triester phosphite andtriester and diester phosphate compounds of the formula: ##STR5##wherein:

i is 0 or 1 (where i=0 indicates phosphite, and i=1 indicatesphosphate);

k equals 1 (where k=0 indicates diester, and k=1 indicates triester)whenever i equals 0, or k equals 0 or 1 whenever i equals 1;

j, R₁, R₂, R₃, and R₄ are as indicated above; and W represents anoligonucleotide, a polymer support, or an oligonucleotide linked to apolymer support. Oligonucleotides include fragments of single-strandedand double-stranded RNA, and fragments of single- and double-strandedDNA. Preferably the linking agents is conjugated to the terminal 5'carbon of an oligonucleotide, the terminal 3' carbon of anoligonucleotide, or the terminal 2' carbon of RNA. More preferably, thelinking agent is conjugated to the terminal 5' carbon of anoligonucleotide, and most preferably the linking agent is conjugated tothe terminal 5' carbon of a fragment of single-stranded DNA.

Polymer supports may have a variety of forms and compositions. Thepolymer support can be derived from naturally occurring materials,naturally occurring materials which are synthetically modified, andsynthetic materials. Of particular interest are polysaccharides,particularly crosslinked polysaccarides, such as agarose, which isavailable as Sepharose, dextran, available as Sephadex and Sephacyl,cellulose, starch and the like (Sepharose, Sephadex, and Sephacyl beingtrademarked products of Pharmacia Fine Chemicals). Other materialsinclude polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers ofhydroxyethyl methacrylate and methyl methacrylate, silicones, teflonsglasses, cells, or the like. In addition to solid supports in the formof particles and the like, the polymer support may also be in the formof liquid particles comprising a lipophilic or amphiphilic membrane,which serves to contain an internal fluid and define a space. Suchparticles include vesicles, cells, and liposomes. Preferably A'represents an insoluble polymer support having hydroxyl functionalities.The linking agents of the invention are attached to polymer supportshaving hydroxyl functionalities by following the procedures generallydescribed below for attaching the linking agents to oligonucleotides.

The bracket on the lefthand side of Formula V (enclosing H and R₁)indicates that this embodiment includes both the protected anddeprotected forms of the compound.

Oligonucleotides are linked to ploymer supports by standard techniquesof affinity chromatography or, for example, by linking means disclosedby Caruthers et al. in U.S. Pat. Nos. 4,458,066 and 4,415,732, or thelike.

The triester phosphite and triester and diester phosphate conjugates ofthe present invention further include compounds of the formula: ##STR6##wherein i, k, s, t, R₁, R₄, and W are as indicated above.

Generally the triester phosphate compounds of the invention are readilyobtained from the above-defined phosphite conjugates by oxidation, e.g.with the use of I₂ in water, 2,6-lutidine and tetrahydrofuran. Oxidationis extremely rapid (1-2 minutes).

The diester phosphate conjugates of the invention are readily obtainedfrom the above-defined triester phosphates by standard techniques, forexample when R₄ is methyl, the diester phosphates are obtained from thetriester phosphates by treatment with thiophenol/triethylamine for about30 minutes. The general procedure for synthesizing the phosphoramiditeprecursors of Formulas III and IV comprises the following steps.Halo-substituted-N,N-di-substituted-O-substituted phosphine, defined bythe formula: ##STR7## wherein X is a halogen, usually chloro, and R',R", and R₄ are as indicated above, is reacted with an amino-protectedalcohol amine defined by the formula: ##STR8## wherein R₁, R₂, and R₃are as indicated above, in an aprotic solvent, such as dichloromethane,or the like, containing a non-nucleophilic base, for example atrialkylamine, such as N,N-diisopropylethyl amine, or the like, whichabsorbs the halogen acid released during the reaction. Preferably thereaction takes place under an inert atmosphere, such as argon. Acidconditions in the reaction mixture should be avoided as acid causes theamine of the phosphoramidite product to protonate, and thereby becomereactive. The non-nucleophilic base reduces the likelihood of sidereactions between the base and activated phosphoramidites.

Whenever the alkyl moiety, i.e. --(CR₂ R₃)_(n) --, of theamino-protected alcohol amine is cycloalkyl, e.g. as in Formula IV, theamide or carbamate moiety of the alcohol amine is preferably in the cisconfiguration with the hydroxy; otherwise, formation of theoxazaphosphaheterocycle becomes unlikely, or even impossible, because ofthe spatial separation of the two groups.

After reacting the above materials, the reaction mixture, hereinafterreferred to as the first reaction mixture, is washed with a mildly basicsolution to remove salts of the non-nucleophilic base. Finally, thefirst reaction mixture is dried, e.g. with MgSO₄, Na₂ SO₄, or the like,to give the phosphoramidite precursor.

The heterocycles of Formulas I and II are obtained by heating theappropriate precursor represented by Formulas III or IV, respectively,to form a second reaction mixture, and then separating the heterocyclefrom the mixture. The necessary amount of heating, i.e. temperature andduration, varies with different embodiments of the invention, preferablyheating includes raising the precursor to a temperature within the rangeof about 25° to 250° C., more preferably from about 25° to 150° C., andmost preferably from about 25° to 100° C. The choice of method ofseparation depends on the nature of the substituent groups, R₁, R₂, R₃,and R₄. For example, as a rough approximation when the aggregatemolecular weight of the substituents is sufficiently low, the steps ofheating and separating can be accomplished by distilling. Other methodsof separation include crystallization and chromatography. Preferablyconjugates of oligonucleotides and linking agents of the invention areformed by attaching the linking agent to oligonucleotides synthesized bythe solid phase synthetic methods developed by Caruthers and hiscoworkers, e.g. Caruthers et al., pgs. 1-17, in Genetic Engineering,Vol. 4, Setlow and Hollaender, Eds. (Plenum Press, New York, 1982), andCaruthers et al., U.S. Pat. No. 4,458,066. Attachment of the linkingagent occurs as the final step in the synthetic process; that is, thelinking agent is attached to the oligonucleotide as if it were anucleotide subunit in the Caruthers et al. method.

The following examples serve to illustrate the present invention. Theconcentrations of reagents, temperatures, and values of other variableparameters are only to exemplify the application of the presentinvention and are not to be considered as limitations thereof.

EXAMPLE I Synthesis of the phosphoramidite precursor of2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane

Chloro-N,N-diisopropylaminomethoxy phosphine (5.0 ml, available formAldrich Chemical Co., Milwaukee, Wis.) was added dropwise at 0° C. to astirred solution of N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide (3.9 g)and diisopropylethylamine (5.0 ml) in dichloromethane (about 40 ml)under argon. (N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide is synthesizedfollowing the procedures disclosed by Lazarus and Benkovic in J. Am.Chem. Soc., Vol. 101, pgs. 4300-4312 (1979): Ethyl trifluoroacetate(56.8 g, 0.4 mol) in 50 mL of chloroform is added dropwise to a stirredsolution of 24.4 g (0.4 mol) of ethanolamine in 50 mL of chloroform. Thesolution is stirred at room temperature for 5 h, rotary evaporated toremove the solvent, and distilled at 115° C. (4.3 Torr) to give 58.5 gof oil that crystallized upon scratching.) After stirring at roomtemperature for 0.5 hours the reaction mixture was washed twice with0.2M potassium carbonate solution and once with brine, dried (MgSO₄ ),and concentrated under reduced pressure to giveN-(2-(N',N'-diisopropylaminomethoxyphosphinyloxy)ethyl)-2,2,2-trifluoroacetamideas a colorless liquid (7.77 g).

¹ H-NMR (CD₂ Cl₂): δ3.6 (6H, m), 3.4 (3H, d, J=12), 1.2 (12H, d, J=6.5)

³¹ P-NMR (CD₂ Cl₂, ¹ H-decoupled): δ149(s)

EXAMPLE II Synthesis of the phosphoramidite precursor of2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclohexane

Chloro-N,N-diisopropylaminomethoxy phosphine (3.7 ml) was added dropwiseat 0° C. to a stirred solution ofN-(3-hydroxypropyl)-2,2,2-trifluoroacetamide (2.9 g, synthesized from3-amino-1-propanol and ethyltrifluoroacetate in a manner similar to thatdisclosed by Lazarus and Benkovic, J. Amer. Chem. Soc., Vol. 101,pgs.4300-4312 (1979)) and diisopropylethylamine (3.7 ml) indichloromethane (about 20 ml) under argon. After stirring at roomtemperature for 3 hours, the reaction mixture was poured into hexane(100 ml) and stirred. The mixture was allowed to settle and thesupernatant was separated and concentrated under reduced pressure togiveN-(3-(N',N'-diisopropylaminomethoxyphosphinyloxy)propyl)-2,2,2-trifluoroacetamideas a colorless liquid (5.2 g).

³¹ P-NMR (CH₂ Cl₂, ¹ H decoupled): δ149 (s)

EXAMPLE III Synthesis of2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane

N-(2-(N',N'-diisopropylaminomethoxyphosphinyloxy)ethyl)-2,2,2-trifluoroacetamide(7.7 g) was distilled (58°-59° C. at 0.8 Torr) to quantitatively yield2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane as acolorless liquid.

IR (film): 1705, 1420, 1230, 1200, 1160, 1020, 965 cm⁻¹

¹ H-NMR (CD₂ Cl₂): δ4.45 (2H, m), 3.65 (2H, m), 3.60 (3H, d, J=12)

³¹ P-NMR (CD₂ Cl₂, ¹ H decoupled): δ132(s), 125 (q, J=61)

MS: m/e 217 (M⁺), 197, 148, 136, 123, 120, 109, 92, 79, 70(100), 69, 62

EXAMPLE IV Attaching2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane to the 5'terminus of an oligonucleotide

Attachment of 2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentaneto a 5' hydroxyl of an oligonucleotide was performed on an AppliedBiosystems 380A DNA synthesizer (Applied Biosystems, Foster City,Calif.), or comparable instrument. Caruthers et al, U.S. Pat. No.4,458,066; Caruthers et al, U.S. Pat. No. 4,415,732; and Caruthers etal, "New Methods for Synthesizing Deoxyoligonucleotides," in GeneticEngineering, Vol. 4, pgs. 1-17 (Plenum Press, New York, 1982) providedetailed descriptions of the chemistry used by the Applied Biosystems380A DNA synthesizer. Accordingly, these references are incorporated byreference for those descriptions.2-Methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane was used as a0.2M acetonitrile solution in combination with 0.5Mtetrazole/acetonitrile solution to form an activated reagent in thesynthesis cycle. The normal synthesizer cycle was modified only duringthe addition of the activated reagent in the following manner. Theactivated reagent was added twice with 1 hour wait times after eachaddition. The coupling yields were about 95%. Normal deprotection withthiophenol/triethylamine and then ammonium hydroxide gave a5'-aminoethylphosphate oligonucleotide. Similar yields were obtainedwhen the activated reagent comprised an acetonitrile solution containing0.2M 2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane and 0.1M4-dimethylaminopyridine. In this case the modified activator reagent wasadded once, and allowed to react for about 15 minutes.

EXAMPLE V Attaching2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane to the 3'terminus of an oligonucleotide

Attachment is accomplished in substantially the same manner as describedin Example IV, except the oligonucleotide is synthesized in the 3'direction in accordance with the procedure generally described inCaruthers et al, U.S. Pat. No. 4,458,066. (Roughly the difference isthat the oligonucleotide is synthesized from 5'N,N-diisopropylaminophosphoramidites of 3'-protected nucleosides insteadof 3' N,N-diisopropylaminophosphoramidites of 5'-protected nucleotides.Alternatively, the oligonucleotide is synthesized in the 3' directionusing the phosphotriester method of Khorana and Itakura (i.e., Khorana,Science, Vol. 203, pgs. 614-625 (1979); Itakura et al. J. Biol. Chem.,Vol. 250, pgs. 4592-4600, both of these references being incorporated byreference), or its modification by others, for example Letsinger andMahaderan, J. Am. Chem. Soc., Vol. 187, pgs. 3526- (1965). In any casethe linking agent is attached as a final addition in place ofnucleotide.

EXAMPLE VI Attaching Fluorescein isothiocyanate (FITC) to a 5'aminoethylphosphate oligonucleotide

A dimethylformamide solution of fluorescein-6-isothiocyanate (25microliters at a concentration of 10 mg/ml, e.g. available formMolecular Probes, Inc., Junction City, Oreg.) was added to a solution of5'-aminoethylphosphate TCCCAGTCACGACGTT (0.020 micromole, unpurifiedmaterial being made on an Applied Biosystems 380A DNA synthesizer; hereT=thymidine, C=cytidine, G=guanosine, and A=adenosine) in water (200microliters) and 1M NaHCO₃ /Na₂ CO₃ buffer, pH 9.0 (25 microliters). Theresulting solution was stored in the dark at room temperature for atleast 6 hours. To remove the unconjugated dye, the reaction mixture waspassed through an equilibrated 10 ml Sephadex (trademark of PharmaciaFine Chemicals) G-25 (medium) column with water. The band of coloredmaterial in the excluded volume was collected. The crude 5'-fluoresceinaminoethylphosphate oligonucleotide was purified by HPLC (e.g.Perkin-Elmer Series 4, or comparable instrument) on a Vydac C18 column(No. 213TP54), or the like, in a linear gradient of 10-20%acetonitrile/0.1M triethylammonium acetate, pH 7.0.

We claim:
 1. A compound of the formula: ##STR9## wherein: j is in the range of 2 to 10;R₁ is an amino protection group; R₂ and R₃ taken separately each represent hydrogen; alkyl having from 1 to 6 carbon atoms; halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted alkyl having from 1 to 6 carbon atoms; cycloalkyl having from 3 to 6 carbon atoms; cyano, halo, or nitro; R₄ is alkyl, alkenyl, aryl, aralkyl, or cycloalkyl containing up to 10 carbon atoms; and R' and R" taken separately each represent alkyl, aryl, aralkyl, cycloalkyl, or cycloalkylalkyl containing up to 10 carbon atoms; or R' and R" taken together form an alkylene chain containing up to 5 carbon atoms in the principal chain and a total of up to 10 carbon atoms with both terminal valence bonds of said chain being attached to the nitrogen atom to which R' and R" are attached; or R' and R" when taken together with the nitrogen atom to which they are attached from a hydrogen-saturated nitrogen heterocycle of five or six atoms selected from the group consisting of carbon, nitrogen, oxygen, and sulfur.
 2. A compound according to claim 1 wherein j is in the range of 2 to
 4. 3. A compound according to claim 2 wherein j is in the range of 2 to
 3. 4. A compound according to claim 3 wherein R₁ taken together with the nitrogen to which it is attached represents a base-labile amide or carbamate protection group, and R₄ is alkyl having from 1 to 6 carbon atoms; halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-, beta-substituted ethyl; halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted phenyl; or halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted phenylethyl.
 5. A compound according to claim 4 wherein R₄ is alkyl having from 1 to 6 carbon atoms; trihalomethyl-, cyano-, nitro-, or sulfo-beta-substituted ethyl; halo-,cyano-, nitro-, or sulfo-substituted phenyl; or halo-, cyano-, nitro-, or sulfo-substituted phenylethyl.
 6. A compound according to claim 5 wherein R₄ is methyl, 4-nitrophenylethyl, or beta-cyanoethyl.
 7. A compound according to claim 4 wherein R₁ is trihaloacetyl, acetoacetyl, or fluorenylmethyl carbamate.
 8. A compound according to claim 7 wherein R₄ is alkyl having from 1 to 6 carbon atoms; trihalomethyl-, cyano-, nitro-, or sulfo-beta-substituted ethyl; halo-, cyano-, nitro-, or sulfo-substituted phenyl; or halo-, cyano-, nitro-, or sulfo-substituted phenylethyl.
 9. A compound according to claim 8 wherein R₄ is methyl, 4-nitrophenylethyl, or beta-cyanoethyl.
 10. A compound according to claim 9 wherein R₁ is trifluoroacetyl, acetoacetyl, 9-(2-sulfo)fluorenylmethyl carbamate, or 9-fluorenylmethyl carbamate.
 11. A compound according to claim 10 wherein R' and R" taken separately are alkyls selected from the group consisting of isopropyl, t-butyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, sec-pentyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, neohexyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, and 1-ethyl-2-methylpropyl, or R' and R" taken together with the nitrogen atom to which they are attached is morpholino, pyrrolidino, or piperidino.
 12. A compound according to claim 11 wherein R' and R" taken separately are isopropyls, or R' and R" taken together is morpholino.
 13. A compound according to claim 12 wherein R₂ and R₃ are hydrogens.
 14. A compound according to claim 3 wherein R₁ is an acid-labile amino protection group.
 15. A compound according to claim 14 wherein:R₁ is trityl or alkoxy-substituted trityl, wherein said alkoxy substituent has from 1 to 6 carbon atoms; R₄ is alkyl having from 1 to 6 carbon atoms; cyano-, trihalomethyl-, nitro-, or sulfo-beta-substituted ethyl; halo-, cyano-, nitro-, or sulfo-substituted phenyl; or halo-, cyano-, nitro-, or sulfo-substituted phenylethyl; and R' and R" taken separately are alkyls selected from the group consisting of isopropyl, t-butyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, sec-pentyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, neohexyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, and 1-ethyl-2-methylpropyl; or R' and R" taken together with the nitrogen atom to which they are attached is morpholino, pyrrolidino, or piperidino.
 16. A compound according to claim 15 wherein R₁ is 4-monomethoxytrityl or 4,4'-dimethoxytrityl.
 17. A cycloalkane of the formula: ##STR10## the cycloalkane having a protected amino substituent and a phosphoramidite substituent wherein:s is in the range of 0 to 8, t is in the range of 0 to 8, and s+t is in the range of 1 to 8; R₁ is an amino protection group; R₄ is alkyl, alkenyl, aralkyl, or cycloalkyl containing up to 10 carbon atoms; and R' and R" taken separately are alkyls selected from the group consisting of isopropyl, t-butyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, sec-pentyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, neohexyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, and 1-ethyl-2-methylpropyl; or R' and R" taken together with the nitrogen atom to which they are attached is morpholino, pyrrolidino, or piperidino.
 18. A compound according to claim 17 wherein s is in the range of 1 to 3, t is in the range of 0 to 2, and the protected amine and phosphoramidite substituents to the cycloalkane defined by said formula are in cis configuration.
 19. A compound according to claim 18 wherein R₁ taken together with the nitrogen atom to which it is attached is a base-labile amide or carbamate protection group, and R₄ is alkyl having from 1 to 6 carbon atoms, halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-beta-substituted ethyl; halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted phenyl; or halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted phenylethyl.
 20. A compound according to claim 19 wherein R₁ is trihaloacetyl, acetoacetyl, or fluorenylmethyl carbamate.
 21. A compound according to claim 20 wherein:t is 0; s is in the range of 1 to 2; R₁ is trifluoroacetyl or acetoacetyl; R₄ is methyl, 4-nitrophenylethyl, or beta-cyanoethyl; and R' and R" taken separately are isopropyl; or R' and R" taken together with the nitrogen atom to which they are attached is morpholino.
 22. A compound according to claim 18 wherein R₁ is an acid-labile amino protection group.
 23. A compound according to claim 22 wherein:R₄ is alkyl having from 1 to 6 carbon atoms; halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-beta-substituted ethyl; halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted phenyl; or halo-, cyano-, nitro-, sulfo-, halomethyl-, dihalomethyl-, or trihalomethyl-substituted phenylethyl; and R' and R" taken separately are alkyls selected from the group consisting of isopropyl, t-butyl, sec-butyl, neopentyl, tert-pentyl, isopentyl, sec-pentyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, neohexyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, and 1-ethyl-2-methylpropyl; or R' and R" taken together with the nitrogen atom to which they are attached are morpholino, pyrrolidino, or piperidino.
 24. A compound according to claim 23 wherein t is 0, s is in the range of 1 to 2, and R₁ is trityl or alkoxy-substituted trityl, wherein said alkoxy substituent has from 1 to 6 carbon atoms.
 25. A compound according to claim 24 wherein R₁ is 4-monomethoxytrityl or 4,4'-dimethoxytrityl. 